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murine lung epithelial cell line mle 12 crl 2110 (ATCC)

Name: murine lung epithelial cell line mle 12 crl 2110
Brand: ATCC
Rating: 98 (1042 reviews)

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ATCC murine lung epithelial cell line mle 12 crl 2110
Murine Lung Epithelial Cell Line Mle 12 Crl 2110, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1042 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Concentration‐response analysis of cAMP elevating agents on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to cAMP elevating agent (a and b) forskolin and (c and d) isoproterenol in the basolateral (bottom) compartment for 3 h at various concentrations. (a and c) A dose–response curve for measured apical fluid pH is depicted and (b and d) comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4–5, FSK; n = 5, ISO). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Concentration‐response analysis of cAMP elevating agents on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to cAMP elevating agent (a and b) forskolin and (c and d) isoproterenol in the basolateral (bottom) compartment for 3 h at various concentrations. (a and c) A dose–response curve for measured apical fluid pH is depicted and (b and d) comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4–5, FSK; n = 5, ISO). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001.

Article Snippet: The human airway epithelial cell line Calu‐3 (male, age 25) (ATCC, HTB‐55), derived from lung adenocarcinoma tissue, was cultured in Alpha‐Minimum Essential medium (α‐MEM) (Corning, 10‐022‐CV) supplemented with 10% fetal bovine serum (VWR, 080‐450), 1% HEPES (Corning, 25‐060‐CI), and 1% penicillin–streptomycin (VWR, 97063‐708) at 37°C and 5% CO 2 for both submerged and air‐liquid interface (ALI) culture conditions.

Techniques: Concentration Assay

Control experiment in Ringer's solution to validate drug‐induced pH changes in the absence of cells. Various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) were administered to the basolateral (bottom) compartment for 3 h to evaluate the change in pH in the absence of human airway epithelial (Calu‐3) cells. Data presented as means ± SD ( n = 3). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Control experiment in Ringer's solution to validate drug‐induced pH changes in the absence of cells. Various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) were administered to the basolateral (bottom) compartment for 3 h to evaluate the change in pH in the absence of human airway epithelial (Calu‐3) cells. Data presented as means ± SD ( n = 3). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Techniques: Control

Effect of various pharmacological interventions on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 7). A two‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001; #### p ≤ 0.0001.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of various pharmacological interventions on apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to various combinations of cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM), CFTR potentiator VX‐770 (1 μM), PDE‐4 inhibitor roflumilast (1 μM), and ABCC4 inhibitor MK‐571 (10 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 7). A two‐way ANOVA with subsequent multiple comparisons was used for statistical analysis. **** p ≤ 0.0001; #### p ≤ 0.0001.

Techniques:

CFTR inhibitors administered in the apical or basolateral compartment do not impact apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to CFTR inhibitors (a and c) CFTRinh‐172 and (b and d) GlyH‐101 in the apical (a and b, top) or basolateral (c and d, bottom) compartment for 3 h at various concentrations. cAMP elevating agent forskolin (0.1 μM) was used as a positive control and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 5). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: CFTR inhibitors administered in the apical or basolateral compartment do not impact apical fluid pH. Human airway epithelial (Calu‐3) cells were exposed to CFTR inhibitors (a and c) CFTRinh‐172 and (b and d) GlyH‐101 in the apical (a and b, top) or basolateral (c and d, bottom) compartment for 3 h at various concentrations. cAMP elevating agent forskolin (0.1 μM) was used as a positive control and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 5). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Techniques: Positive Control

Effect of cAMP elevating agents FSK and ISO post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of cAMP elevating agents FSK and ISO post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with cAMP elevating agents forskolin (0.1 μM) and isoproterenol (0.01 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Techniques: Inhibition

Effect of CFTR potentiator VX‐770 post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with CFTR potentiator VX‐770 (1 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Journal: Physiological Reports

Article Title: Effects of cAMP and CFTR modulation on apical fluid pH in human airway Calu‐3 cells

doi: 10.14814/phy2.70747

Figure Lengend Snippet: Effect of CFTR potentiator VX‐770 post‐CFTR inhibition on apical fluid pH. Human airway epithelial (Calu‐3) cells were pre‐treated with CFTR inhibitors (a) CFTRinh‐172 (10 μM) and (b) GlyH‐101 (10 μM) to the apical (top) compartment for 30 min prior to treatment with CFTR potentiator VX‐770 (1 μM) to the basolateral (bottom) compartment for 3 h. Measured apical fluid pH is depicted and comparisons between treatment groups were performed. Data presented as means ± SD ( n = 4). A one‐way ANOVA with subsequent multiple comparisons was used for statistical analysis.

Techniques: Inhibition

a . Schematic drawing of the transwell-platform. Serum deprived NMuMG cells were covered with an agarose disk followed by a metal load of defined weight, exerting uniaxial compressive force. At the indicated times, the load was removed, and the cells were harvested for different assays. b . NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through 8 µm transwell pores. Transmigrated cells adhering to the bottom of the 6-well plate form colonies, which were stained with crystal violet. The colony-forming units were quantified to compare the migratory potential across conditions. N=18; unpaired t test comparison; p<0.0001****; p<0.05 considered significant. c . and d . Serum deprived NMuMG cells invading the empty space were imaged by phalloidin staining 24 hours after removing the inset of the ibidi chambers, in the presence (+) or absence (-) of compressive pressure. Scale bar: 50 μm ( c ). Note that compressed cells acquire an elongated, migratory-like phenotype, due to a profound rearrangement of their cytoskeleton. The roughness of the edge was quantified ( d ) as described in . The normalized wound edge perimeter was plotted as a metric of the compressive stress phenotype. Unpaired t-test comparison; p<0.0001****; p<0.05 considered significant. e , f and g . NMuMG cells under normal (-) or compressive stress (+) conditions were stained with DAPI (grey and blue) or rhodamine-phalloidin (grey and green), to visualize nuclei or the actin cytoskeleton, respectively ( e ). Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Scale bar: 3 μm. The nuclear ( f ) and cell ( g ) shape changes were quantified as an increase in the cell or nuclear aspect ratio after cellpose-based cell or nuclear segmentation. N=11 and N=8, respectively; Unpaired t-test comparison; p<0.0001****; p<0.05, considered significant. h . The adherens junction marker E-cadherin (gray and green) and the tight junction protein ZO1 (gray and magenta) were immunostained in compressed (+) and non-compressed (-) NMuMG cells. Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Note that upon compression, E-cadherin is internalized and forms punctate structures, while ZO1 disappears from tight junctions.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a . Schematic drawing of the transwell-platform. Serum deprived NMuMG cells were covered with an agarose disk followed by a metal load of defined weight, exerting uniaxial compressive force. At the indicated times, the load was removed, and the cells were harvested for different assays. b . NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through 8 µm transwell pores. Transmigrated cells adhering to the bottom of the 6-well plate form colonies, which were stained with crystal violet. The colony-forming units were quantified to compare the migratory potential across conditions. N=18; unpaired t test comparison; p<0.0001****; p<0.05 considered significant. c . and d . Serum deprived NMuMG cells invading the empty space were imaged by phalloidin staining 24 hours after removing the inset of the ibidi chambers, in the presence (+) or absence (-) of compressive pressure. Scale bar: 50 μm ( c ). Note that compressed cells acquire an elongated, migratory-like phenotype, due to a profound rearrangement of their cytoskeleton. The roughness of the edge was quantified ( d ) as described in . The normalized wound edge perimeter was plotted as a metric of the compressive stress phenotype. Unpaired t-test comparison; p<0.0001****; p<0.05 considered significant. e , f and g . NMuMG cells under normal (-) or compressive stress (+) conditions were stained with DAPI (grey and blue) or rhodamine-phalloidin (grey and green), to visualize nuclei or the actin cytoskeleton, respectively ( e ). Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Scale bar: 3 μm. The nuclear ( f ) and cell ( g ) shape changes were quantified as an increase in the cell or nuclear aspect ratio after cellpose-based cell or nuclear segmentation. N=11 and N=8, respectively; Unpaired t-test comparison; p<0.0001****; p<0.05, considered significant. h . The adherens junction marker E-cadherin (gray and green) and the tight junction protein ZO1 (gray and magenta) were immunostained in compressed (+) and non-compressed (-) NMuMG cells. Scale bar: 20 μm. The inset magnifies a section of cells at the epithelia edge. Note that upon compression, E-cadherin is internalized and forms punctate structures, while ZO1 disappears from tight junctions.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Comparison, Marker

a. Experimental outline of compressive stress application on the transwell platform. b. NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through narrow 8 µm transwell pores. Transmigrated cells adhere to the bottom of the 6-well plate form colonies, which were stained with crystal violet. Representative images of colonies are shown. c. Wound edge roughness quantification. Images of the tissue edges obtained with ibidi chambers are converted to a mask image, and the perimeter of the edge is divided by a straight line connecting two edge points. d. Schematic workflow to quantify the nuclear and cell morphological changes.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Experimental outline of compressive stress application on the transwell platform. b. NMuMG cells were exposed (+) or not (-) to compression and allowed to migrate for 24 hours through narrow 8 µm transwell pores. Transmigrated cells adhere to the bottom of the 6-well plate form colonies, which were stained with crystal violet. Representative images of colonies are shown. c. Wound edge roughness quantification. Images of the tissue edges obtained with ibidi chambers are converted to a mask image, and the perimeter of the edge is divided by a straight line connecting two edge points. d. Schematic workflow to quantify the nuclear and cell morphological changes.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining

a. Schematic representation of the ERK5 MAPK pathway. ERK5 is phosphorylated and thereby activated by MEK5 on the TY residues in its T-loop. Active ERK5 phosphorylates cytoplasmic targets, and translocates into the nucleus, where it binds and phosphorylates the transcription factor MEF2. In the reporter construct, active MEF2 drives expression of GFP ( MEF2p -GFP). b. NMuMG cells were reverse-transfected with the MEF2p -GFP reporter plasmid and either an empty control vector (EV) or a vector expressing an HA-tagged dominant-active MEK5 mutant (HA-MEK5DD). GFP expression was monitored with (+) or without (-) compressive stress after 24 hours by western blot analysis. Where indicated (+), XMD8-92 was added to inhibit ERK5 activity. α-tubulin is used as a loading control. The numbers below represent densitometric quantification of GFP level across conditions, normalized to the tubulin loading control and relative to the empty control vector in the absence of compression and ERK5 inhibition. c. Western blot (WB) analysis confirmed elevated levels of active ERK5 (p-ERK5) after 3 hours of compression in serum-starved NMuMG cells, while the total ERK5 levels remained unchanged. α-tubulin controls equal loading. The numbers below represent the densitometric quantification of p-ERK5 and ERK5 levels across conditions normalized to GAPDH levels and relative to control condition without compression and ERK5 depletion. d - f. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with a control siRNA oligo (siCTR) were exposed (+) or not (-) to compressive stress for 3 hours and the localization of p-ERK5 was analyzed by immunofluorescence analysis. Representative images show increased p-ERK5 at cell-cell contacts ( d ). Scale bar: 20 μm. Total- ( e ) and epithelial ( f ) p-ERK5 fluorescence intensity was quantified, and statistical significance confirmed by an ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05 was considered statistically significant.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Schematic representation of the ERK5 MAPK pathway. ERK5 is phosphorylated and thereby activated by MEK5 on the TY residues in its T-loop. Active ERK5 phosphorylates cytoplasmic targets, and translocates into the nucleus, where it binds and phosphorylates the transcription factor MEF2. In the reporter construct, active MEF2 drives expression of GFP ( MEF2p -GFP). b. NMuMG cells were reverse-transfected with the MEF2p -GFP reporter plasmid and either an empty control vector (EV) or a vector expressing an HA-tagged dominant-active MEK5 mutant (HA-MEK5DD). GFP expression was monitored with (+) or without (-) compressive stress after 24 hours by western blot analysis. Where indicated (+), XMD8-92 was added to inhibit ERK5 activity. α-tubulin is used as a loading control. The numbers below represent densitometric quantification of GFP level across conditions, normalized to the tubulin loading control and relative to the empty control vector in the absence of compression and ERK5 inhibition. c. Western blot (WB) analysis confirmed elevated levels of active ERK5 (p-ERK5) after 3 hours of compression in serum-starved NMuMG cells, while the total ERK5 levels remained unchanged. α-tubulin controls equal loading. The numbers below represent the densitometric quantification of p-ERK5 and ERK5 levels across conditions normalized to GAPDH levels and relative to control condition without compression and ERK5 depletion. d - f. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with a control siRNA oligo (siCTR) were exposed (+) or not (-) to compressive stress for 3 hours and the localization of p-ERK5 was analyzed by immunofluorescence analysis. Representative images show increased p-ERK5 at cell-cell contacts ( d ). Scale bar: 20 μm. Total- ( e ) and epithelial ( f ) p-ERK5 fluorescence intensity was quantified, and statistical significance confirmed by an ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05 was considered statistically significant.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Construct, Expressing, Transfection, Plasmid Preparation, Control, Mutagenesis, Western Blot, Activity Assay, Inhibition, Immunofluorescence, Fluorescence, Comparison

a. NMuMG cells siRNA depleted for ERK5 (siERK5) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression and allowed to migrate for 24 hours through 8 μm transwell pores. Colonies formed by transmigrated cells were visualized by crystal violet staining and quantified as described in . N=6; ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05, considered statistically significant. b - d. Invading NMuMG cells growing in ibidi chambers, either siRNA-depleted for ERK5 or treated with control oligos ( b, c ) or exposed to the ERK5 inhibitor XMD8-92 or DMSO solvent control ( d ), were visualized by phalloidin staining in the presence (+) or absence (-) of compressive pressure for 24 hours. The wound edge roughness was quantified to compare the different conditions. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. e. and f. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector expressing dominant-active HA-MEK5DD. Where indicated, the ERK5 inhibitor XMD8-92 or solvent control (DMSO) was added. The wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=3; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g - i. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with control oligos (siCTR) exposed (+) or not (-) to compressive force conditions were stained with DAPI, rhodamine-phalloidin, and immunostained with antibodies against ZO1 to visualize tight junctions. Scale bar: 20 μm. The cell shape changes ( h ) and nuclear aspect ratio ( i ) were quantified as described in the legend to and , respectively. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. NMuMG cells siRNA depleted for ERK5 (siERK5) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression and allowed to migrate for 24 hours through 8 μm transwell pores. Colonies formed by transmigrated cells were visualized by crystal violet staining and quantified as described in . N=6; ANOVA followed by Tukey multiple comparison test; ***p<0.001; p<0.05, considered statistically significant. b - d. Invading NMuMG cells growing in ibidi chambers, either siRNA-depleted for ERK5 or treated with control oligos ( b, c ) or exposed to the ERK5 inhibitor XMD8-92 or DMSO solvent control ( d ), were visualized by phalloidin staining in the presence (+) or absence (-) of compressive pressure for 24 hours. The wound edge roughness was quantified to compare the different conditions. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. e. and f. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector expressing dominant-active HA-MEK5DD. Where indicated, the ERK5 inhibitor XMD8-92 or solvent control (DMSO) was added. The wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=3; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g - i. NMuMG cells siRNA-depleted for ERK5 (siERK5) or treated with control oligos (siCTR) exposed (+) or not (-) to compressive force conditions were stained with DAPI, rhodamine-phalloidin, and immunostained with antibodies against ZO1 to visualize tight junctions. Scale bar: 20 μm. The cell shape changes ( h ) and nuclear aspect ratio ( i ) were quantified as described in the legend to and , respectively. N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Comparison, Solvent, Transfection, Plasmid Preparation, Expressing

a. and b . crystal violet staining of transmigrated NMuMG colonies exposed (+) or not (-) to compression and treated with siRNA controls (siCTR) or oligos targeting ERK5 (siERK5) ( a ), or upon ERK5 inhibition with XMD8-92 or the DMSO-solvent control ( b ). c. Quantification of colony forming units (CFU) of b . N=8; Statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test; ***p<0.001; p<0.05, considered significant. d. Invading NMuMG cells growing in ibidi chambers treated with XMD8-92 or for control with DMSO, were exposed (+) or not (-) to compression. After 24 hours, the cells were stained with phalloidin and filamentous actin visualized by microscopy. Scale bar: 50 μm. e. Representative images of NMuMG cells with (+) or without (-) compression were stained with DAPI to visualize the nuclei and phalloidin to probe filamentous actin. As indicated, the cells were treated with XMD8-92 or DMSO for control. Scale bar: 20 μm. f. Cell shape changes were quantified as the increase in the cell aspect ratio after Cellpose-based cell segmentation. N=4; ANOVA and Tukey’s multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g. Morphological changes of nuclei are quantified as the increase in nuclear aspect ratio after Cellpose-based nuclear segmentation. N=4; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note that upon compression, ERK5 activity is required for actin rearrangement and cell-cell contact dissolution.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. and b . crystal violet staining of transmigrated NMuMG colonies exposed (+) or not (-) to compression and treated with siRNA controls (siCTR) or oligos targeting ERK5 (siERK5) ( a ), or upon ERK5 inhibition with XMD8-92 or the DMSO-solvent control ( b ). c. Quantification of colony forming units (CFU) of b . N=8; Statistical analysis was performed using ANOVA followed by Tukey’s multiple comparison test; ***p<0.001; p<0.05, considered significant. d. Invading NMuMG cells growing in ibidi chambers treated with XMD8-92 or for control with DMSO, were exposed (+) or not (-) to compression. After 24 hours, the cells were stained with phalloidin and filamentous actin visualized by microscopy. Scale bar: 50 μm. e. Representative images of NMuMG cells with (+) or without (-) compression were stained with DAPI to visualize the nuclei and phalloidin to probe filamentous actin. As indicated, the cells were treated with XMD8-92 or DMSO for control. Scale bar: 20 μm. f. Cell shape changes were quantified as the increase in the cell aspect ratio after Cellpose-based cell segmentation. N=4; ANOVA and Tukey’s multiple comparison; ***p<0.001; p<0.05, considered statistically significant. g. Morphological changes of nuclei are quantified as the increase in nuclear aspect ratio after Cellpose-based nuclear segmentation. N=4; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note that upon compression, ERK5 activity is required for actin rearrangement and cell-cell contact dissolution.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Inhibition, Solvent, Control, Comparison, Microscopy, Activity Assay, Dissolution

a – c. NMuMG cells exposed (+) or not (-) to compression were treated with XMD8-92 or for control with DMSO ( a , c ), or with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) ( b ). Cells were then stained with phalloidin to visualize filamentous actin and antibodies against E-cadherin ( a , b ) or ZO1 ( c ). Scale bar: 20 μm. ERK5 activity is required for compression-induced dissolution of tight and adherens junctions as well as E-cadherin internalization. d. Confocal images of actin and ZO1 in NMuMG cells transfected with a control vector or a plasmid overexpressing HA-MEK5DD to constitutively activate ERK5. As indicated the cells were treated with the ERK5 inhibitor XMD8-92 or for control DMSO. Scale bar: 20 μm. Note that HA-MEK5DD overexpression promotes an invasive morphology even in the absence of compression.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a – c. NMuMG cells exposed (+) or not (-) to compression were treated with XMD8-92 or for control with DMSO ( a , c ), or with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) ( b ). Cells were then stained with phalloidin to visualize filamentous actin and antibodies against E-cadherin ( a , b ) or ZO1 ( c ). Scale bar: 20 μm. ERK5 activity is required for compression-induced dissolution of tight and adherens junctions as well as E-cadherin internalization. d. Confocal images of actin and ZO1 in NMuMG cells transfected with a control vector or a plasmid overexpressing HA-MEK5DD to constitutively activate ERK5. As indicated the cells were treated with the ERK5 inhibitor XMD8-92 or for control DMSO. Scale bar: 20 μm. Note that HA-MEK5DD overexpression promotes an invasive morphology even in the absence of compression.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Activity Assay, Dissolution, Transfection, Plasmid Preparation, Over Expression

a. Experimental workflow used for phospho-proteomic profiling of NMuMG cells to classify compression-regulated, ERK5-dependent and independent phospho-sites. Phospho-peptides were identified by MS-analysis after F e -NTA enrichment, using extracts prepared from serum-starved NMuMG cells exposed (filled circles) or not (open circles) to compression for 3 hours, with or without ERK5 inhibition by XMD8-92. b. - e. Volcano plot showing differential protein phosphorylation in compression versus no-compression comparison (p-adj. ≤ 0.05, log2FC>1.0) marking ERK5-independent ( b ) and ERK5-dependent ( c ) compression-induced phosphosites. Proteins involved in cell-to-cell contact and the regulation of the actin cytoskeleton ( c ) and gene expression regulation and nuclear import ( b ) are highlighted, respectively. Each phosphorylated protein is represented by its most significant phosphopeptide. The p-value <0.05 is considered significant and corresponds to an ANOVA two-sided p-value, adjusted for multiple testing across the cells-treatment conditions of interest after post hoc analysis (FDR). Gene ontology (GO) enrichment analysis revealed ERK5-dependent targets related to cell-cell junction and actin cytoskeleton organization ( e ), and pressure-induced, but ERK5-independent targets related to nuclear transport regulation and post-transcriptional gene silencing ( d ). Bonferroni- adjusted p-value < 0.05 corresponds to two-sided Fisher’s exact test. All genes in Mus musculus database are used as reference list. f. Experimental workflow to functionally validate candidate ERK5-substrates. Candidates were siRNA depleted in serum starved NMuMG cells growing in ibidi chambers and examined by the epithelial edge assay after 24 hours of compression. The screen was repeated twice, each time dividing the candidates into five or six batches, with each batch including non-targeting siRNAs and siERK5 controls. Cells were fixed and stained with rhodamine phalloidin and the edge roughness imaged and quantified. The resulting phenotypes were grouped in 4 functional categories, which are illustrated by the edge images of NMuMG cells exposed (+) or not (-) to compressive pressure g . Category 1: siRNA knockdown has no influence on the phenotypic response to compression; category 2: siRNA knockdown prevents the response to compression; category 3: siRNA knockdown increases cell dissociation upon compression; category 4: siRNA knockdown leads to cell dissociation even in the absence of compression. h. and i. Graphic comparison of wound edge roughness of two screen replicates. The wound edge roughness of each target is normalized to the average value of the control siRNAs and log2 transformed. The dashed lines indicate 95% confidence intervals (95% CI) around the median of all measurements in each independent screen. Candidates that significantly reduce the wound edge perimeter upon compression compared to cells treated with control siRNAs are marked in blue. Candidates enhancing the wound edge roughness with ( i ) or without compression ( h ) are marked in red and green, respectively.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Experimental workflow used for phospho-proteomic profiling of NMuMG cells to classify compression-regulated, ERK5-dependent and independent phospho-sites. Phospho-peptides were identified by MS-analysis after F e -NTA enrichment, using extracts prepared from serum-starved NMuMG cells exposed (filled circles) or not (open circles) to compression for 3 hours, with or without ERK5 inhibition by XMD8-92. b. - e. Volcano plot showing differential protein phosphorylation in compression versus no-compression comparison (p-adj. ≤ 0.05, log2FC>1.0) marking ERK5-independent ( b ) and ERK5-dependent ( c ) compression-induced phosphosites. Proteins involved in cell-to-cell contact and the regulation of the actin cytoskeleton ( c ) and gene expression regulation and nuclear import ( b ) are highlighted, respectively. Each phosphorylated protein is represented by its most significant phosphopeptide. The p-value <0.05 is considered significant and corresponds to an ANOVA two-sided p-value, adjusted for multiple testing across the cells-treatment conditions of interest after post hoc analysis (FDR). Gene ontology (GO) enrichment analysis revealed ERK5-dependent targets related to cell-cell junction and actin cytoskeleton organization ( e ), and pressure-induced, but ERK5-independent targets related to nuclear transport regulation and post-transcriptional gene silencing ( d ). Bonferroni- adjusted p-value < 0.05 corresponds to two-sided Fisher’s exact test. All genes in Mus musculus database are used as reference list. f. Experimental workflow to functionally validate candidate ERK5-substrates. Candidates were siRNA depleted in serum starved NMuMG cells growing in ibidi chambers and examined by the epithelial edge assay after 24 hours of compression. The screen was repeated twice, each time dividing the candidates into five or six batches, with each batch including non-targeting siRNAs and siERK5 controls. Cells were fixed and stained with rhodamine phalloidin and the edge roughness imaged and quantified. The resulting phenotypes were grouped in 4 functional categories, which are illustrated by the edge images of NMuMG cells exposed (+) or not (-) to compressive pressure g . Category 1: siRNA knockdown has no influence on the phenotypic response to compression; category 2: siRNA knockdown prevents the response to compression; category 3: siRNA knockdown increases cell dissociation upon compression; category 4: siRNA knockdown leads to cell dissociation even in the absence of compression. h. and i. Graphic comparison of wound edge roughness of two screen replicates. The wound edge roughness of each target is normalized to the average value of the control siRNAs and log2 transformed. The dashed lines indicate 95% confidence intervals (95% CI) around the median of all measurements in each independent screen. Candidates that significantly reduce the wound edge perimeter upon compression compared to cells treated with control siRNAs are marked in blue. Candidates enhancing the wound edge roughness with ( i ) or without compression ( h ) are marked in red and green, respectively.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Inhibition, Phospho-proteomics, Comparison, Gene Expression, Staining, Functional Assay, Knockdown, Control, Transformation Assay

a. Schematic illustration of the ERK5 signaling pathway phosphorylating S909 of the myosin light chain phosphatase subunit MYPT1, which in turn dephosphorylates and thereby activates the myosin light chain (MLC). Active myosin controls cortical tension by pulling on actin fibers. b . and c . NMuMG cells siRNA depleted for ERK5 (siERK5), MYPT1 (siMYPT1) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression for 3 hours, and immunostained for phospho-MLC (p-MLC). Note that MLC phosphorylation is decreased upon compression by an ERK5 and MYPT1-dependent mechanism. Scale bar: 20 μm ( b ). Fluorescence intensity of p-MLC at cell edges was quantified as described for phospho-ERK5. Compression significantly reduces epithelial p-MLC levels while ERK5 depletion protects p-MLC at cell-cell contacts ( c ). d. Extracts prepared from cells expressing HA-tagged ERK5 (HA-ERK5) or carrying an empty control plasmid (HA-empty) were incubated with HA-affinity resins. Immobilized proteins were incubated with a peptide encompassing the S909 phosphorylation site of MYPT1 (S909-WT) or a mutant peptide where Ser909 was replaced by an alanine residue (S909-A) in the presence of γ- 32 P-ATP. The peptides were separated by thin layer chromatography (TLC) and analyzed by autoradiography. The numbers below represent the densitometric quantification of phosphorylated peptides relative to active ERK5 upon compression. e. - g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note the elongated morphology and significantly weaker intercellular connections in NMuMG cells expressing Ser909-D compared to Ser909-WT and Ser909-A mutants ( g ).

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. Schematic illustration of the ERK5 signaling pathway phosphorylating S909 of the myosin light chain phosphatase subunit MYPT1, which in turn dephosphorylates and thereby activates the myosin light chain (MLC). Active myosin controls cortical tension by pulling on actin fibers. b . and c . NMuMG cells siRNA depleted for ERK5 (siERK5), MYPT1 (siMYPT1) or treated with a control oligo (siCTR) were exposed (+) or not (-) to physical compression for 3 hours, and immunostained for phospho-MLC (p-MLC). Note that MLC phosphorylation is decreased upon compression by an ERK5 and MYPT1-dependent mechanism. Scale bar: 20 μm ( b ). Fluorescence intensity of p-MLC at cell edges was quantified as described for phospho-ERK5. Compression significantly reduces epithelial p-MLC levels while ERK5 depletion protects p-MLC at cell-cell contacts ( c ). d. Extracts prepared from cells expressing HA-tagged ERK5 (HA-ERK5) or carrying an empty control plasmid (HA-empty) were incubated with HA-affinity resins. Immobilized proteins were incubated with a peptide encompassing the S909 phosphorylation site of MYPT1 (S909-WT) or a mutant peptide where Ser909 was replaced by an alanine residue (S909-A) in the presence of γ- 32 P-ATP. The peptides were separated by thin layer chromatography (TLC) and analyzed by autoradiography. The numbers below represent the densitometric quantification of phosphorylated peptides relative to active ERK5 upon compression. e. - g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Wound edge roughness was visualized by phalloidin staining ( e ) and quantified to compare the different conditions ( f ). N=5; ANOVA with Tukey multiple comparison; ***p<0.001; p<0.05, considered statistically significant. Note the elongated morphology and significantly weaker intercellular connections in NMuMG cells expressing Ser909-D compared to Ser909-WT and Ser909-A mutants ( g ).

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Phospho-proteomics, Fluorescence, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Residue, Thin Layer Chromatography, Autoradiography, Transfection, Staining, Comparison

a . – c : NMuMG cells exposed (+) or not (-) to compression were treated with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) or MYPT1 (siMYPT1). Cells were stained for total MLC ( a ), non-muscle myosin IIA heavy chain ( b ), or the cell tension marker vinculin ( c ), and visualized by immunofluorescence. Scale bar: 20 µm. d. Quantification Ser909 phosphorylation of MYPT1 using PRM analysis. NMuMG cells were compressed in the presence or absence of XMD8-92 (inhibitor). N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. e. Analysis of total MYPT1 levels in NMuMG cells compressed in the presence or absence of XMD8-92 (inhibitor) using phosphoproteomic analysis. N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. f. HA-tagged ERK5 was immobilized on HA-beads and incubated in the presence of γ 32 P-ATP with a peptide encompassing the Ser909 phosphorylation site of MYPT1, either as wild-type (S909-WT) or the S909 alanine mutant (S909-A). As indicated, the in vitro kinase assay was performed in the presence of XMD8-92 or DMSO for control. g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing myc-tagged wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Expression levels were visualized by immunoblotting with myc-antibody (upper blot). An antibody against tubulin (Tub) controls equal loading. While Ser909-A is overexpressed, Ser909-WT and Ser909-D are present at comparable levels.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a . – c : NMuMG cells exposed (+) or not (-) to compression were treated with siRNA controls (siCTR) or sioligos targeting ERK5 (siERK5) or MYPT1 (siMYPT1). Cells were stained for total MLC ( a ), non-muscle myosin IIA heavy chain ( b ), or the cell tension marker vinculin ( c ), and visualized by immunofluorescence. Scale bar: 20 µm. d. Quantification Ser909 phosphorylation of MYPT1 using PRM analysis. NMuMG cells were compressed in the presence or absence of XMD8-92 (inhibitor). N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. e. Analysis of total MYPT1 levels in NMuMG cells compressed in the presence or absence of XMD8-92 (inhibitor) using phosphoproteomic analysis. N=3; One-tailed t-test; ***p<0.001; p<0.05, considered statistically significant. f. HA-tagged ERK5 was immobilized on HA-beads and incubated in the presence of γ 32 P-ATP with a peptide encompassing the Ser909 phosphorylation site of MYPT1, either as wild-type (S909-WT) or the S909 alanine mutant (S909-A). As indicated, the in vitro kinase assay was performed in the presence of XMD8-92 or DMSO for control. g. NMuMG cells grown in ibidi chambers were reverse-transfected with an empty control vector or a vector overexpressing myc-tagged wild-type MYPT1 (Ser909-WT), or MYPT1 mutants changing S909 either to a phosphomimicking glutamic acid (Ser909-D) or a non-phosphorylatable alanine (Ser909-A) residue. Expression levels were visualized by immunoblotting with myc-antibody (upper blot). An antibody against tubulin (Tub) controls equal loading. While Ser909-A is overexpressed, Ser909-WT and Ser909-D are present at comparable levels.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Staining, Marker, Immunofluorescence, Phospho-proteomics, One-tailed Test, Incubation, Mutagenesis, In Vitro, Kinase Assay, Control, Transfection, Plasmid Preparation, Residue, Expressing, Western Blot

a. NMuMG cells siRNA depleted for ROCK (siROCK), ERK5 (siERK5), or both together (siROCK + siERK5), or treated with a control oligo (siCTR), were exposed (+) or not (-) to physical compression for 3 hours, and phospho-threonine 852 (T852) of MYPT1 (p-MYPT1) was analyzed by immunofluorescent staining. Scale bar: 20 μm. Note that the ROCK-dependent T852 phosphorylation is decreased upon compression. b. and c. NMuMG cells grown in ibidi chambers were treated with the ROCK inhibitor Y27632, its solvent DMSO, or together with the ERK5 inhibitor XMD8-92. Cells were exposed (+) or not (-) to compressive pressure for 24 hours, and wound edge roughness was quantified after phalloidin staining. Note that ROCK and ERK5 activities antagonize each other. N=5; ANOVA followed by post hoc Bonferroni multiple comparison of relevant conditions; ***p<0.001; p<0.05 considered statistically significant. d . During unstressed conditions, ROCK phosphorylates MYPT1 on its inhibitory Thr852 site (Thr853 in human), thereby increasing active myosin (phospho-MLC) and stabilizing actin structures and cell-cell junctions. Compressive stress inhibits ROCK but activates ERK5, which then phosphorylates MYPT1 on Ser909. This phosphorylation switch activates the myosin light chain phosphatase complex (MLCP), leading to myosin inhibition and in turn changes in cortical actin structures and disruption of cell-cell junctions, thus promoting an invasive behavior.

Journal: bioRxiv

Article Title: Compressive mechanical stress activates ERK5 to regulate cortical tension and promote invasive cellular traits

doi: 10.64898/2026.01.30.702739

Figure Lengend Snippet: a. NMuMG cells siRNA depleted for ROCK (siROCK), ERK5 (siERK5), or both together (siROCK + siERK5), or treated with a control oligo (siCTR), were exposed (+) or not (-) to physical compression for 3 hours, and phospho-threonine 852 (T852) of MYPT1 (p-MYPT1) was analyzed by immunofluorescent staining. Scale bar: 20 μm. Note that the ROCK-dependent T852 phosphorylation is decreased upon compression. b. and c. NMuMG cells grown in ibidi chambers were treated with the ROCK inhibitor Y27632, its solvent DMSO, or together with the ERK5 inhibitor XMD8-92. Cells were exposed (+) or not (-) to compressive pressure for 24 hours, and wound edge roughness was quantified after phalloidin staining. Note that ROCK and ERK5 activities antagonize each other. N=5; ANOVA followed by post hoc Bonferroni multiple comparison of relevant conditions; ***p<0.001; p<0.05 considered statistically significant. d . During unstressed conditions, ROCK phosphorylates MYPT1 on its inhibitory Thr852 site (Thr853 in human), thereby increasing active myosin (phospho-MLC) and stabilizing actin structures and cell-cell junctions. Compressive stress inhibits ROCK but activates ERK5, which then phosphorylates MYPT1 on Ser909. This phosphorylation switch activates the myosin light chain phosphatase complex (MLCP), leading to myosin inhibition and in turn changes in cortical actin structures and disruption of cell-cell junctions, thus promoting an invasive behavior.

Article Snippet: Murine mammary gland epithelial cell line NMuMG, was obtained from the laboratory of G. Christofori and originally from American Type Culture Collection (ATCC).

Techniques: Control, Staining, Phospho-proteomics, Solvent, Comparison, Inhibition, Disruption

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